Melatonin synthesis in retina: cAMP-dependent transcriptional regulation of chicken arylalkylamine N-acetyltransferase (Aanat) by a CRE-like sequence (CLS) and a TTATT repeat motif in the proximal promoter

• Authors: Rashidul Haque, Emory University; Nelson W. Chong, University of Leicester; Fatima Ali, Emory University; Shyam S. Chaurasia, Emory University; Trisha Sengupta, Emory University; Eugene Chun, Emory University; Jennifer Christina Howell, Emory University; David C. Klein, National Institutes of Health; P Michael Iuvone, Emory University
• Journal Title: Journal of Neurochemistry
• Volume: Volume 119, Number 1
• Publisher: Wiley: 12 months | 2011-10, Pages 6-17
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1111/j.1471-4159.2011.07397.x
• Subjects: Health Sciences, Opthamology; Biology, Neuroscience
• Keywords: CRE; AP1; cAMP; CREB; circadian rhythm; arylalkylamine N-acetyltransferase; Aanat; gene regulation; retina; melatonin; JunD; cFos

Abstract

Arylalkylamine N-acetyltransferase (AANAT) is the key regulatory enzyme controlling the daily rhythm of melatonin biosynthesis. In chicken retinal photoreceptor cells, Aanat transcription and AANAT activity are regulated in part by cAMP-dependent mechanisms. The purpose of this study was to identify regulatory elements within the chicken Aanat promoter responsible for cAMP-dependent induction. Photoreceptor-enriched retinal cell cultures were transfected with a luciferase reporter construct containing up to 4kb of 5′-flanking region and the first exon of Aanat. Forskolin treatment stimulated luciferase activity driven by the ~4kb promoter construct and by all 5′-deletion constructs except the smallest, Aanat (−217 to +120)luc. Maximal basal and forskolin-stimulated expression levels were generated by the Aanat (−484 to +120)luc construct. This construct lacks a canonical cyclic AMP-response element (CRE), but contains two other potentially important elements in its sequence: an eight times TTATT repeat (TTATT8) and a CRE-like sequence (CLS). Electrophoretic mobility shift assays (EMSA), luciferase reporter assays, chromatin immunoprecipitation, and siRNA experiments provide evidence that these elements bind c-Fos, JunD, and CREB to enhance basal and forskolin-stimulated Aanat transcription. We propose that the CLS and TTATT8 elements in the 484 bp proximal promoter interact to mediate cAMP-dependent transcriptional regulation of Aanat.